In silico characterization of ligand-receptor interactions for U-47700, N,N-didesmethyl-U-47700, U-50488 at mu- and kappa-opioid receptors.

The increasing misuse of novel synthetic opioids (NSOs) represents a serious public health concern. In this regard, U-47700 (trans-3,4-dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methylbenzamide) and related "U-compounds" emerged on recreational drug markets as synthetic substitutes for illicit heroin and constituents of counterfeit pain medications. While the pharmacology of U-compounds has been investigated using in vitro and in vivo methods, there is still a lack of understanding about the details of ligand-receptor interactions at the molecular level. To this end, we have developed a molecular modeling protocol based on docking and molecular dynamics simulations to assess the nature of ligand-receptor interactions for U-47700, N,N-didesmethyl U-47700, and U-50488 at the mu-opioid receptor (MOR) and kappa-opioid receptor (KOR). The evaluation of ligand-receptor and ligand-receptor-membrane interaction energies enabled the identification of subtle conformational shifts in the receptors induced by ligand binding. Interestingly, the removal of two key methyl groups from U-47700, to form N,N-didesmethyl U-47700, caused a loss of hydrogen bond contact with tryptophan (Trp)229, which may underlie the lower interaction energy and reduced MOR affinity for the compound. Taken together, our results are consistent with the reported biological findings for U-compounds and provide a molecular basis for the MOR selectivity of U-47700 and KOR selectivity of U-50488.

κ-Opioid Receptors Improve Vascular Endothelial Dysfunction in Salt-Sensitive Hypertension via PI3K/Akt/eNOS Signaling Pathway.

κ-Opioid receptors (κ-OR) are widely used to regulate the activity of the cardiovascular system. To explore the effect and mechanism of κ-OR on salt-sensitive hypertensive endothelial dysfunction, we used Dah1 rats to construct a rat model of salt-sensitive hypertension on a high-salt (HS) diet. Then, the rats were treated with κ-OR activators U50,488H (1.25 mg/kg) and inhibitor nor-BNI (2.0 mg/kg) for 4 weeks, respectively. The rat aortas were collected to detect the contents of NO, ET-1, AngII, NOS, T-AOC, SO, and NT. Protein expression was determined for NOS, Akt, and Caveolin-1. In addition, the vascular endothelial cells were extracted, and the levels of NO, TNF-α, IL-1, IL-6, IL-8, IL-10, p-Akt, and p-eNOS in cell supernatants were detected. In vivo results showed that compared with the HS group, treated with U50,488H promoted rats' vasodilation by increasing the NO content and decreasing ET-1 and AngII contents. U50,488H reduced endothelial cell apoptosis and attenuated vascular, smooth muscle cell and endothelial cell injury. U50,488H also enhanced the rats' response to oxidative stress by increasing the NOS and T-AOC contents. Moreover, U50,488H increased the eNOS, p-eNOS, Akt, and p-AKT expression and decreased the iNOS and Caveolin-1 expression. In vitro results showed that U50,488H promoted NO, IL-10, p-Akt, and p-eNOS levels in endothelial cell supernatants versus the HS group. And U50,488H reduced the adhesion of peripheral blood mononuclear cells and polymorphonuclear neutrophils to endothelial cells and the migration function of polymorphonuclear neutrophils. Our study suggested that κ-OR activation may improve vascular endothelial dysfunction in salt-sensitive hypertensive rats through the PI3K/Akt/eNOS signaling pathway. This may be a potential therapeutic approach in the treatment of hypertension.

Inhibition of P2X7 receptors by Lu AF27139 diminishes colonic hypersensitivity and CNS prostanoid levels in a rat model of visceral pain.

Visceral pain is a prominent feature of various gastrointestinal diseases. The P2X7 receptor is expressed by multiple cell types including dorsal root ganglion satellite glial cells, macrophages, and spinal microglia, all of which have been implicated in nociceptive sensitization. We have used the selective and CNS penetrant P2X7 receptor antagonist Lu AF27139 to explore this receptor's role in distinct rat models of inflammatory and visceral hypersensitivity. Rats injected with CFA in the hindpaw displayed a marked reduction in hindpaw mechanical threshold, which was dose-dependently reversed by Lu AF27139 (3-30 mg/kg, p.o.). In rats injected with TNBS in the proximal colon, the colorectal distension threshold measured distally was significantly lower than sham treated rats at 7 days post-injection (P < 0.001), indicative of a marked central sensitization. Colonic hypersensitivity was also reversed by Lu AF27139 (10-100 mg/kg) and by the κ-opioid receptor agonist U-50,488H (3 mg/kg, s.c.). Moreover, both Lu AF27139 and U-50,488H prevented a TNBS-induced increase in spinal and brain levels of PGE2 and LTB4, as well as an increase in brain levels of PGF2α and TXB2. Lu AF27139 was well tolerated as revealed by a lack of significant effect on rotarod motor function and coordination at all doses tested up to 300 mg/kg. Thus, P2X7 receptor antagonism is efficacious in a rat model of visceral pain, via a mechanism which potentially involves attenuation of microglial function within spinal and/or supraspinal pain circuits, albeit a peripheral site of action cannot be excluded.

A κ-OR Agonist Protects the Endothelial Function Impaired by Hyperuricemia Through Regulating the Akt/eNOS Signal Pathway.

To investigate the effects of κ-OR agonist on hyperuricemia rats and injured endothelial function, as well as the underlying mechanism. A hyperuricemia model was established on rats. The endothelial protective effects of U50,488H were evaluated and compared to the controlled groups. The protein levels of eNOS, p-eNOS, Akt, and p-Akt were determined using western blot analysis. ELISA was employed to measure the expression of soluble ET-1, ICAM-1, TNF-α, and NO in cell supernatants and rat serum samples. Cell migration and the artery tension were determined by in vitro functional assays. The suppressed production of ET-1, ICAM-1, and NO in the hyperuricemia rats was promoted by the treatment of U50,488H, which was reversed by the co-administration of nor-BNI. P-eNOS/eNOS and p-Akt/Akt were up-regulated by the incubation of serum from hyperuricemia rats, which was down-regulated by the introduction of U50,488H. The vascular tension of vessels incubated with U50,488H was higher than the baseline in the presence of ACh, which was lower than baseline in the presence of SNAP. U50,488H significantly promoted the release of ET-1, ICAM-1, and NO, and inhibited the release of TNF-α from endothelial cells and the migration ability of neutrophils in the presence of hyperuricemia rat serum, which were reversed by the co-incubation with nor-BNI, Akt inhibitor or L-NAME. U50,488H protected the endothelial function impaired by hyperuricemia through regulating the Akt/eNOS signal pathway.

Opioid receptor activation modulates the calcium current in the cochlear outer hair cells of the rat.

Previous works showed that opioid peptides are produced by olivocochlear efferent neurons, while cochlear hair cells express opioid receptors. It has been proposed that opioids protect the auditory system from damage by intense stimulation, although their use for therapeutic or illicit purposes links to hearing impairment. Therefore, it is relevant to study the effect of opioids in the auditory system to define their functional expression and mechanism of action. This study investigated the modulation of the Ca2+ currents by opioid peptides in the rat outer hair cells (OHC) using the whole-cell patch-clamp technique. The influence of agonists of the three opioid receptor subtypes (µ, δ, and κ) was studied. The κ opioid receptor agonist U-50488 inhibits the Ca2+ currents in a partially reversible form. Coincidently, norbinaltorphimine (a κ receptor antagonist) blocked the U-50488 inhibitory effect on the Ca2+ current. The δ and the µ opioid receptor agonists did not significantly affect the Ca2+ currents. These results indicate that the κ opioid receptor activation inhibits the Ca2+ current in OHC, modulating the intracellular Ca2+ concentration when OHCs depolarize. The modulation of the auditory function by opioids constitutes a relevant mechanism with a potential role in the physiopathology of auditory disturbances.

Pharmacological activation of kappa opioid receptors in the nucleus accumbens core and ventral tegmental area increases the aversive effects of nicotine.

Aversive effects of nicotine play an important role in the development of nicotine dependence. However, neural substrates and/or brain regions that play a role in the aversive effects of nicotine have not been fully identified. Previous work done in our laboratory showed that systemic administration of kappa opioid receptors (KORs) agonist ±U50488 increased the aversive effects of nicotine. In this study, we assessed the effects of KOR activation in specific brain regions, namely, the nucleus accumbens (NAcc) core and ventral tegmental area (VTA) on the aversive effects of nicotine using the conditioned taste aversion model. Separate groups of Wistar rats were implanted with cannulae above either the NAcc core or the VTA. KOR agonist (±U50488) was bilaterally infused in the NAcc core (0, 0.3, and 3 ug/0.5 ul/side) or VTA (0, 0.3, 1.5, and 3 ug/0.5 ul/side) prior to receiving nicotine (0.4 mg/kg, base; s.c.) during conditioning. Bilateral infusion of the KOR agonist (3 ug/0.5 ul/side) in the NAcc core or the VTA increased the aversive effects of nicotine compared with respective saline controls. Together, these results suggest that pharmacological activation of the KORs in the NAcc core and VTA dose dependently modulate the aversive effects of nicotine. Because aversive effects of nicotine determine susceptibility to development of nicotine dependence, we can conclude that KOR activity in the NAcc and VTA after administration of nicotine may determine susceptibility to the development of nicotine dependence.

Agonist-promoted kappa opioid receptor (KOR) phosphorylation has behavioral endpoint-dependent and sex-specific effects.

We reported previously that the selective agonist U50,488H promoted phosphorylation of the mouse kappa opioid receptor (mKOR) in vitro at four residues in the C-terminal domain. In this study, we generated a mutant mouse line in which all the four residues were mutated to Ala (K4A) to examine the in vivo functional significance of agonist-induced KOR phosphorylation. U50,488H promoted KOR phosphorylation in brains of the wildtype (WT), but not K4A, male and female mice. Autoradiography of [3H] 69,593 binding to KOR in brain sections showed that WT and K4A mice had similar KOR distribution and expression levels in brain regions without sex differences. In K4A mice, U50,488H inhibited compound 48/80-induced scratching and attenuated novelty-induced hyperlocomotion to similar extents as in WT mice without sex differences. Interestingly, repeated pretreatment with U50,488H (80 mg/kg, s.c.) resulted in profound tolerance to the anti-scratch effects of U50,488H (5 mg/kg, s.c.) in WT mice of both sexes and female K4A mice, while in male K4A mice tolerance was attenuated. Moreover, U50,488H (2 mg/kg) induced conditioned place aversion (CPA) in WT mice of both sexes and male K4A mice, but not in female K4A mice. In contrast, U50,488H (5 mg/kg) caused CPA in male, but not female, mice, regardless of genotype. Thus, agonist-promoted KOR phosphorylation plays important roles in U50,488H-induced tolerance and CPA in a sex-dependent manner, without affecting acute U50,488H-induced anti-pruritic and hypo-locomotor effects. These results are the first to demonstrate sex differences in the effects of GPCR phosphorylation on the GPCR-mediated behaviors.

Novel selective κ agonists SLL-039 and SLL-1206 produce potent antinociception with fewer sedation and aversion.

SLL-039 (N-cyclopropylmethyl-7α-4'-(N'-benzoyl) amino-phenyl-6,14-endoethano-tetrahydronorthebaine) and SLL-1206 (N-cyclopropylmethyl-7α-3'-(p-methoxybenzyl) amino-phenyl-6,14-endoethano-tetrahydronorthebaine) are two 4,5-epoxymorphinan-based high selective κ receptor agonists that we recently discovered. In the present study we characterized their pharmacological properties in comparison with arylacetamide-based typical κ agonist U50,488H. We showed that both SLL-039 and SLL-1206 produced potent and long-lasting antinociceptive actions in three different rodent models of pain via activation of κ opioid receptor. In hot-plate assay, the antinociceptive potency of SLL-039 and SLL-1206 increased about 11-and 17.3-fold compared to U50,488H and morphine, respectively, with ED50 values of 0.4 mg/kg. Following repeated administration, SLL-1206, SLL-039, and U50,488H all developed analgesic tolerance tested in hot-plate assay. U50,488H and SLL-039 produced antipruritic effects in a dose-dependent manner, whereas SLL-1206 displayed some antipruritic effects only at very low doses. In addition, SLL-1206 was capable of decreasing morphine-induced physical dependence. More importantly, SLL-039 and SLL-1206 at effective analgesic doses did not cause sedation and conditioned place aversion (CPA), whereas U50,488H did. In comparison with SLL-039, SLL-1206 caused similar antinociceptive responses, but fewer sedation and CPA. In conclusion, our results suggest that SLL-039 and SLL-1206 have potential to be developed as novel analgesic agents, and 4,5-expoxymorphinan scaffold is an attractive structure for the development of selective κ agonists with fewer side effects.

Oxytocin Is a Positive Allosteric Modulator of κ-Opioid Receptors but Not δ-Opioid Receptors in the G Protein Signaling Pathway.

Oxytocin (OT) influences various physiological functions such as uterine contractions, maternal/social behavior, and analgesia. Opioid signaling pathways are involved in one of the analgesic mechanisms of OT. We previously showed that OT acts as a positive allosteric modulator (PAM) and enhances µ-opioid receptor (MOR) activity. In this study, which focused on other opioid receptor (OR) subtypes, we investigated whether OT influences opioid signaling pathways as a PAM for δ-OR (DOR) or κ-OR (KOR) using human embryonic kidney-293 cells expressing human DOR or KOR, respectively. The CellKeyTM results showed that OT enhanced impedance induced by endogenous/exogenous KOR agonists on KOR-expressing cells. OT did not affect DOR activity induced by endogenous/exogenous DOR agonists. OT potentiated the KOR agonist-induced Gi/o protein-mediated decrease in intracellular cAMP, but did not affect the increase in KOR internalization caused by the KOR agonists dynorphin A and (-)-U-50488 hydrochloride (U50488). OT did not bind to KOR orthosteric binding sites and did not affect the binding affinities of dynorphin A and U50488 for KOR. These results suggest that OT is a PAM of KOR and MOR and enhances G protein signaling without affecting ß-arrestin signaling. Thus, OT has potential as a specific signaling-biased PAM of KOR.

Quantification of kappa opioid receptor ligand potency, efficacy and desensitization using a real-time membrane potential assay.

We explored the utility of the real-time FLIPR Membrane Potential (FMP) assay as a method to assess kappa opioid receptor (KOR)-induced hyperpolarization. The FMP Blue dye was used to measure fluorescent signals reflecting changes in membrane potential in KOR expressing CHO (CHO-KOR) cells. Treatment of CHO-KOR cells with kappa agonists U50,488 or dynorphin [Dyn (1-13)NH2] produced rapid and concentration-dependent decreases in FMP Blue fluorescence reflecting membrane hyperpolarization. Both the nonselective opioid antagonist naloxone and the κ-selective antagonists nor-binaltorphimine (nor-BNI) and zyklophin produced rightward shifts in the U50,488 concentration-response curves, consistent with competitive antagonism of the KOR mediated response. The decrease in fluorescent emission produced by U50,488 was blocked by overnight pertussis toxin pretreatment, indicating the requirement for PTX-sensitive G proteins in the KOR mediated response. We directly compared the potency of U50,488 and Dyn (1-13)NH2 in the FMP and [35S]GTPγS binding assays, and found that both were approximately 10 times more potent in the cellular fluorescence assay. The maximum responses of both U50,488 and Dyn (1-13)NH2 declined following repeated additions, reflecting receptor desensitization. We assessed the efficacy and potency of structurally distinct KOR small molecule and peptide ligands. The FMP assay reliably detected both partial agonists and stereoselectivity. Using KOR-selective peptides with varying efficacies, we found that the FMP assay allowed high throughput quantification of peptide efficacy. These data demonstrate that the FMP assay is a sensitive method for assessing κ-opioid receptor induced hyperpolarization, and represents a useful approach for quantification of potency, efficacy and desensitization of KOR ligands.

κ Opioid Receptor Agonist Inhibits Myocardial Injury in Heart Failure Rats through Activating Nrf2/HO-1 Pathway and Regulating Ca2+-SERCA2a.

OBJECTIVES: We aimed to observe the protective effect of κ opioid receptor (κ-OR) agonist on myocardial injury in heart failure (HF) rats and its effect on Ca2+-SERCA2a and to explore the regulatory mechanism with the Nrf2/HO-1 signaling pathway. METHODS: 50 Sprague-Dawley rats were randomly divided into the following groups: the sham operation group (sham group), HF model group (HF group), HF+κ-OR agonist U50488 group (HU group), HF+U50488H+novel calmodulin-dependent protein kinase II (CaMKII) agonist (oleic acid) (HUO group), and HF+U50488H+Nrf2 inhibitor (HUM group). The HF rat's model was established through surgical ligation of the left anterior descending coronary artery and the exhausting swimming exercise. After that, rat's cardiac function was monitored by echocardiography. HE and MASSON staining was used to detect the myocardial injury, and TUNEL staining was used to detect the myocardial apoptosis. ELISA was performed to detect the biomarkers of oxidative stress. Moreover, the distribution of reactive oxygen species (ROS) and Nrf2 was detected under immunofluorescence. The expression of sarco/endoplasmic reticulum calcium (Ca2+) ATPase (SERCA) 2a, calmodulin, endoplasmic reticulum stress- (ERS-) related proteins, and Nrf2/HO-1 signaling pathway-related proteins were detected by Western Blotting. RESULTS: κ-OR agonist U50488H can significantly enhance rat's cardiac function, reduce the injury and apoptosis of myocardial cells, and alleviate endoplasmic reticulum stress injury in HF rats via upregulating the SERCA2a expression and inhibiting the Ca2+ influx. Furthermore, U50488H could also inhibit the phosphorylation of CaMKII and cAMP-response element binding protein (CREB). Additionally, administration of CaMKII-specific agonist could partially block the therapeutic effect of κ-OR agonist on the myocardium of HF rats. Interestingly, the antagonist of Nrf2 could also significantly reverse the therapeutic effect of κ-OR agonist. Therefore, these results suggested that the effect of U50488H on HF rats is dependent on regulating CaMKII phosphorylation and activating the Nrf2/HO-1 pathway. CONCLUSION: κ-OR agonists U50488H can improve ERS in cardiomyocytes and relieve myocardial injury in HF rats through activating the Nrf2/HO-1 pathway and regulating Ca2+-SERCA2a to inhibit Ca2+ influx.

Activation of κ-opioid receptor inhibits inflammatory response induced by sodium palmitate in human umbilical vein endothelial cells.

OBJECTIVES: The current study aims to investigate the effect of κ-opioid receptor (κ-OR) activation on sodium palmitate (SP)-induced human umbilical vein endothelial cells (HUVECs) inflammatory response and elucidate the underlying mechanisms. METHODS: A hyperlipidemic cell model was established and treated with κ-OR agonist (U50,488H), and antagonist (norbinaltorphimine, nor-BNI), or inhibitors targeting PI3K, Akt or eNOS (LY294002, MK2206-2HCl or L-NAME, respectively). Furthermore, the expression levels of NLRP3, caspase-1, p-Akt, Akt, p-eNOS, and total eNOS were evaluated. Additionally, the production of reactive oxygen species, and levels of inflammatory factors, such as TNF-α, IL-1ß, IL-6, IL-1 and adhesion molecules, such as ICAM-1, VCAM-1, P-selectin, and E-selectin were determined. The adherence rates of the neutrophils and monocytes were assessed as well. RESULTS: The SP-induced hyperlipidemic cell model demonstrated increased expression of NLRP3 and caspase-1 proteins (P < 0.05) and elevated ROS levels (P < 0.01), and decreased phosphorylated-Akt and phosphorylated-eNOS expression (P < 0.05). In addition, SP significantly increased TNF-α, IL-1ß, IL-6, ICAM-1, VCAM-1, P-selectin, and E-selectin levels (P < 0.01), decreased IL-10 levels (P < 0.01), and increased the adhesion rates of monocytes and neutrophils (P < 0.01). The SP-induced inflammatory response in HUVECs was ameliorated by κ-OR agonist, U50,488H. However, the protective effect of U50,488H was abolished by κ-OR antagonist, nor-BNI, and inhibitors of PI3K, Akt and eNOS. CONCLUSION: Our findings suggest that κ-OR activation inhibits SP-induced inflammation by activating the PI3K/Akt/eNOS signaling pathway.

Antidepressant-like effects of dezocine in mice: involvement of 5-HT1A and κ opioid receptors.

Dezocine is an opioid with low efficacy at µ-opioid and κ-opioid receptors. It also inhibits the reuptake of norepinephrine and serotonin. Dezocine is an effective analgesic against various clinical painful conditions and is widely used in many Asian countries. Given the unique pharmacology of dezocine, the drug may also have antidepressant-like properties. However, no published preclinical study has explored this possibility. This study examined the potential antidepressant-like activity of dezocine in mice. Male ICR mice were used in the forced swimming test, the tail suspension test, the warm water tail withdrawal test and locomotor activity test to test the effects of dezocine (0.3-3.0 mg/kg). The 5-HT1A receptor antagonist WAY-100635 (1 mg/kg), the µ-opioid receptor antagonist ß-funaltrexamine (2 mg/kg) and the κ-opioid receptor agonist U50488 (1 mg/kg) were also studied in combination with dezocine. Dezocine produced a dose-dependent decrease in the immobility time in the forced swimming test and tail suspension test at doses that did not alter the motoric activity as determined in the locomotion test. WAY-100635 and U50488 but not ß-funaltrexamine pretreatment significantly blocked the effects of dezocine. Dezocine dose-dependently increased the latency in the tail withdrawal test which was blocked by WAY-100635 and ß-funaltrexamine. Combined, these results suggest that dezocine may have antidepressant-like effects. Considering the well-documented analgesic property of dezocine, it may be useful to treat pain and depression comorbidity.

Kappa opioid receptor modulation of endometriosis pain in mice.

The kappa opioid receptor is a constituent of the endogenous opioid analgesia system widely expressed in somatosensory nervous pathways and also in endometrial tissues. This work investigates the possible involvement of kappa opioid receptor on the nociceptive, behavioral and histopathological manifestations of endometriosis in a murine model. Female mice receiving endometrial implants develop a persistent mechanical hypersensitivity in the pelvic area that is stronger during the estrus phase of the estrous cycle. The kappa opioid receptor agonist U50,488H produces a dose-dependent relief of this mechanical hypersensitivity, regardless of the cycle phase. Repeated exposure to a low dose of U50,488H (1 mg/kg/day s.c. for one month) provides sustained relief of mechanical hypersensitivity, without tolerance development or sedative side effects. Interestingly, this treatment also inhibits a decreased rearing behavior associated with spontaneous pain or discomfort in endometriosis mice. This KOR-mediated pain relief does not prevent the anxiety-like behavior or the cognitive impairment exhibited by endometriosis mice, and the growth of endometriotic cysts is also unaltered. These data provide evidence of strong pain-relieving properties of kappa opioid receptor stimulation in female mice with endometriosis pain. The persistence of affective and cognitive manifestations suggests that these comorbidities are independent of pelvic pain and simultaneous treatment of these comorbidities may be necessary for successful management of endometriosis.

Interactive effects of (±)-trans-U50488 and its stereoisomers with cannabinoids.

The adverse effects of mu opioid agonists have spurred a renewed interest in using kappa opioid receptor (KOR) agonists as analgesics. KOR agonists also have potential for development as diuretics for the treatment of edema and hypertension. Here, we evaluated the discriminative stimulus, antinociceptive, and diuretic effects of the kappa agonist (±)-trans-U-50488 and its stereoisomers (-)-(1S,2S)-U-50488 or (+)-(1R,2R)-U-50488) alone and in combination with the cannabinoid agonist (-)-CP 55,940. To establish (±)-U-50488 as a discriminative stimulus, rats (n = 12) were trained to discriminate intraperitoneal (i.p.) administration of 5.6 mg/kg of (±)-trans-U-50488 from saline under a fixed-ratio 20 (FR-20) schedule of food reinforcement. Then, antinociception was assessed using two procedures: warm water tail withdrawal and von Frey paw withdrawal. Diuretic effects were assessed in separate rats (n = 6/group). Doses of (±)-U-50488 and (-)-U-50488 that served as discriminative stimuli produced significant increases in urine output, but at lower doses than those that produced antinociception. In contrast, (+)-U-50488 alone had no discriminative stimulus or diuretic effects at the doses tested, but did produce antinociception in the von Frey assay. When three cannabinoids and morphine were tested in the (±)-U-50488 discrimination procedure to determine the similarity of these drugs' discriminative stimulus effects to those for (±)-U-50488, the rank order similarity was (-)-CP 55,940 > (-)-trans-THC > (+)-WIN 55,212-2 ≥ morphine. (-)-CP 55,940 alone (0.056 mg/kg) partially substituted for the discriminative stimulus effects of (±)-U-50488 and produced significant diuretic and antinociceptive effects. (-)-CP 55,940 in combination with (±)-U-50488 also produced a two-fold leftward shift in the discriminative stimulus curve for (±)-U-50488, and near-additive antinociception with (±)-U-50488 and (+)-U-50488. Further, the diuretic effect of (-)-CP 55,940 was enhanced by a dose of (+)-U50488, which itself did not alter urine output. These data together indicate that a combination of cannabinoid and kappa opioid agonists can enhance diuresis, but may have limited potential for serving as opioid-sparing pharmacotherapeutics for treatment of pain.

Profile of a short-acting κ-antagonist, LY2795050, on self-grooming behaviors, forced swim test and locomotor activity: sex comparison in mice.

BACKGROUND: Novel short-acting κ(kappa)-opioid receptor selective antagonists are translational tools to examine the impact of the κ-receptor/dynorphin system in assays related to central nervous system dysfunction (e.g., substance use disorders, anhedonia and depression). The effects of such compounds have been compared in males and females under very limited conditions. AIMS: The goal of this study was to examine potential sex differences in the effects of a κ-agonist and a short-acting κ-antagonist in an ethologically relevant test of anhedonia, the "splash test" of self-grooming, and also in the forced swim test and in locomotor activity. METHODS: We examined the dose-dependence of grooming deficits caused by the κ-agonist U50,488 (0.1-3.2 mg/kg intraperitoneal (i.p.)) in gonadally intact adult male and female C57BL/6J mice. We then compared the effects of the short-acting κ-antagonist LY2795050 ((3-chloro-4-(4-(((2S)-2-pyridin-3-ylpyrrolidin-1-yl)methyl) phenoxy)benzamide)); 0.032-0.1 mg/kg i.p.) in blocking grooming deficits caused by U50,488 (3.2 mg/kg). The effects of LY2795050 were also studied in the forced swim test (FST). The effects of LY2795050 in blocking the locomotor depressant effects of U50,488 (10 mg/kg) were also studied. RESULTS: U50,488 produced dose-dependent grooming deficits in male and female mice, and LY2795050 prevented these effects. In contrast, LY2795050 decreased immobility in the FST in males at a dose of 0.1 mg/kg, but not in females, up to a dose of 0.32 mg/kg. Also, LY2795050 (0.32 mg/kg) prevented and also reversed the locomotor-depressant effects of U50,488 (10 mg/kg), in males and females. CONCLUSIONS: This study further implicates the κ-receptor system in ethologically relevant aspects of anhedonia, and confirms sexual dimorphism in some behavioral effects of novel κ-antagonists.

κ­opioid receptor agonist U50488H attenuates postoperative cognitive dysfunction of cardiopulmonary bypass rats through the PI3K/AKT/Nrf2/HO­1 pathway.

Postoperative cognitive dysfunction (POCD) is a common complication following cardiopulmonary bypass (CPB). U50488H, a κ­opioid receptor (KOR) agonist, can specifically activate KORs on hippocampal nerve cells, resulting in neuroprotective effects. The present study established a CPB rat model, observed the protective effect of U50488H on CPB­induced POCD and brain damage and explored the regulatory mechanism of the PI3K/AKT/nuclear factor erythroid 2­related factor 2 (Nrf2)/heme oxygenase (HO)­1 pathway. Sprague­Dawley rats were divided into the following groups: Sham operation (Sham group), CPB (CPB group), KOR agonist (U50488H) + CPB (U50488H group), CPB + U50488H + HO­1 antagonist (ZnPP­IX; ZnPP group) and CPB + U50488H + PI3K antagonist (LY294002; LY294002 group), with 10 rats in each group. Neurological scores and the Morris water maze test were used to evaluate cognitive function; hematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP nick end labeling assays were performed to observe hippocampal neuron damage in rats. Immunofluorescence was used to detect reactive oxygen species, glial fibrillary acidic protein and Nrf2 expression in the hippocampus. Enzyme­linked immunosorbent assays were used to detect inflammatory and oxidative stress factors. Western blotting was used to examine the expression of PI3K/AKT/Nrf2/HO­1­related proteins. It was demonstrated that U50488H significantly reduced the neural function score of rats with POCD induced by CPB, relieved cognitive dysfunction, reduced hippocampal neuron damage, inhibited the rate of apoptosis, repaired oxidative stress injury and protected against brain damage caused by CPB. In addition, U50488H could promote Nrf2 entry into the nucleus and upregulate HO­1 and thioredoxin 1 (Trx1) expression. In CPB rats treated with PI3K inhibitors, less Nrf2 was detected in the nucleus and HO­1 and Trx­1 expression levels were reduced in the nucleus. Therefore, U50488H, a KOR agonist, can activate Nrf2/HO­1 via the PI3K/AKT pathway to improve cognitive function and reduce brain damage in CPB rats.

Kappa opioid receptor controls neural stem cell differentiation via a miR-7a/Pax6 dependent pathway.

Although the roles of opioid receptors in neurogenesis have been implicated in previous studies, the mechanism by which κ-opioid receptor (OPRK1) regulates adult neurogenesis remains elusive. We now demonstrate that two agonists of OPRK1, U50,488H and dynorphin A, inhibit adult neurogenesis by hindering neuronal differentiation of mouse hippocampal neural stem cells (NSCs), both in vitro and in vivo. This effect was blocked by nor-binaltorphimine (nor-BNI), a specific antagonist of OPRK1. By examining neurogenesis-related genes, we found that OPRK1 agonists were able to downregulate the expression of Pax6, Neurog2, and NeuroD1 in mouse hippocampal NSCs, in a way that Pax6 regulates the transcription of Neurog2 and Neurod1 by directly interacting with their promoters. Moreover, this effect of OPRK1 was accomplished by inducing expression of miR-7a, a miRNA that specifically targeted Pax6 by direct interaction with its 3'-UTR sequence, and thereby decreased the levels of Pax6, Neurog2, and NeuroD1, thus resulted in hindrance of neuronal differentiation of NSCs. Thus, by modulating Pax6/Neurog2/NeuroD1 activities via upregulation of miR-7a expression, OPRK1 agonists hinder the neuronal differentiation of NSCs and hence inhibit adult neurogenesis in mouse hippocampus.

Kappa-opioid receptor-dependent changes in dopamine and anxiety-like or approach-avoidance behavior occur differentially across the nucleus accumbens shell rostro-caudal axis.

Neural circuit engagement within the nucleus accumbens (NAc) shell is implicated in the regulation of both negative and positive affect. Classically, the dynorphin/kappa opioid receptor (KOR) system in the NAc was believed to promote aversion, while dopamine was viewed as interacting with reward behavior, and KOR activation was known to inhibit dopamine release. Recently, however, both the KOR and dopamine systems have, separately, been shown to have differential effects across the rostro-caudal axis of the NAc shell on hedonic responses. Whether or not this is due to interactions between KORs and dopamine, and if it extends to anxiety-like or approach-avoidance behaviors, remains to be determined. In this study, we examined in rats the relationship between the KOR and dopamine systems in both the rostral and caudal NAc shell using ex vivo fast scan cyclic voltammetry and the impact of KOR activation on affective behavior using exploration-based tasks. We report here that activation of KORs in the caudal NAc shell significantly inhibits dopamine release, stimulates rearing behavior in a novel environment, increases anxiety-like or avoidance behavior, and reduces locomotor activity. In contrast, activation of KORs in the rostral NAc shell inhibits dopamine release to a lesser extent and instead reduces anxiety-like behavior or increases approach behavior. Taken together, these results indicate that there is heterogeneity across the rostro-caudal axis of the NAc shell in the effects of KOR stimulation on affective behaviors, and they suggest that this might be due to differences in KOR control over dopamine release.

Pharmacological and phosphoproteomic approaches to roles of protein kinase C in kappa opioid receptor-mediated effects in mice.

Kappa opioid receptor (KOR) agonists possess adverse dysphoric and psychotomimetic effects, thus limiting their applications as non-addictive anti-pruritic and analgesic agents. Here, we showed that protein kinase C (PKC) inhibition preserved the beneficial antinociceptive and antipruritic effects of KOR agonists, but attenuated the adverse condition placed aversion (CPA), sedation, and motor incoordination in mice. Using a large-scale mass spectrometry-based phosphoproteomics of KOR-mediated signaling in the mouse brain, we observed PKC-dependent modulation of G protein-coupled receptor kinases and Wnt pathways at 5 min; stress signaling, cytoskeleton, mTOR signaling and receptor phosphorylation, including cannabinoid receptor CB1 at 30 min. We further demonstrated that inhibition of CB1 attenuated KOR-mediated CPA. Our results demonstrated the feasibility of in vivo biochemical dissection of signaling pathways that lead to side effects.